OnSite® COVID-19 Ag Rapid Test by CTK Biotech Inc., Poway, CA, USA) on COVID-19 symptomatic patients (n = 380). The overall sensitivity and specificity were 91.0% (95% CI: 84.8–95.3%) and 99.2% (95% CI: 97.1–99.9), against gold standard RT-PCR. The kit was capable of detecting patients even after 06 days of onset of symptoms and the sensitivity can be maximized to 98% in samples with an average RT-PCR Ct ≤ 26.48, demonstrating a high potential of the kit for clinical diagnosis of symptomatic patients in healthcare facilities.Accurate diagnosis at the right moment is the prerequisite for treatment of any disease. Failure to correctly diagnose a disease can result in highly detrimental effects, unmistakably a crucial factor during the COVID-19 pandemic. RT-PCR is the gold standard for COVID-19 detection while there are other test procedures available, such as LAMP, X-Ray, and ELISA. However, these tests are expensive, require sophisticated equipment and a highly trained workforce, and multiple hours or even days are often required to obtain the test results. A rapid and cheap detection system can thus render a solution to the screening system on a larger scale and be added as an aid to the current detection processes. Recently, some rapid antigen-based COVID-19 tests devices have been developed and commercialized. In this study, we evaluated the clinical performance of a new rapid detection device ( Thousands of people are losing their lives every day due to the global COVID-19 pandemic, and fast and accurate diagnosis is still a barrier in most countries . Lack ofSeveral methods have been invented around the globe, including the RT-PCR, loop mediated isothermal amplification (LAMP), enzyme-linked immunosorbent assay (ELISA), rapid diagnostic test (RDT), radiological evaluation, and some mobile applications ,16,17,18An alternative approach would be loop mediated isothermal amplification (LAMP) that like PCR detects the viral nucleic acids, with the advantage of the capacity to test at the field level in remote areas . HoweverOnSite® COVID-19 Ag Rapid Test) for detection of COVID-19 in symptomatic suspected patients in urban population as well as in diagnostic center to evaluate its clinical performance and the prospects for use as a tool for early, rapid, cheap, and efficient diagnosis of COVID-19.All of these aforementioned issues can be avoided by using a rapid and cheap diagnostic method such as the immune-chromatographic test (ICT) in the form of RDT cassette, that can be used in the field level as well as individually, as the test is relatively easy to use, not requiring skilled laboratory professionals. In this study, we performed a prospective evaluation of a new antigen-based lateral flow, qualitative immunoassay device  laboratory services unit (LSU) in Dhaka, Bangladesh for a COVID-19 RT-PCR test between 29 April and 15 September of 2021 were considered for this study. Participants who met the study inclusion criteria (recent fever and two symptoms) were offered to participate in this study. 380 patients with evident clinical COVID-19 symptoms were enrolled in this study for testing by healthcare professionals. The study physician checked and recorded the health status of the participant in a pre-structured form. OnSite COVID-19 Ag Rapid Test, one nasal swab was sample collected by inserting the swab up to 2 cm inside the nostril, following the product’s Instruction for Use (IFU).Both nasopharyngeal and oropharyngeal samples were collected from each study participant and pooled by an expert medical technologist at the designated COVID-19 sample collection booth maintained by the LSU in a single 3 mL Viral transport medium (VTM) for RT-PCR testing. For testing on the ® COVID-19 Ag Rapid Test recognizes SARS-CoV-2 Nucleocapsid Protein. Using ELISA and peptide scanning, the epitopes recognized by the test have been mapped to the N-terminal domain (NTD) of the Nucleocapsid Protein. It is a lateral flow chromatographic immunoassay. The test cassette consists of: (1) a colored conjugate pad containing anti-SARS-CoV-2 antibodies conjugated with colloidal gold (antibody conjugates) and (2) a nitrocellulose membrane strip containing a test line (Ag line) and a control line (C line). The test line is pre-coated with anti-SARS-CoV-2 antibodies, and the C line is pre-coated with control antibodies. The OnSite® COVID Ag rapid test was performed on the spot as per the manufacturer’s IFU. Roughly in this study, a nasal swab is inserted into the nostril until resistance is met at turbinates (less than an inch to nostril) with gentle rotation several times against the nasal wall. The process is repeated in the second nostril to ensure that sample is collected, and the swab is then removed and inserted into an extraction buffer. The extracted sample is then dropped into the sample well of the cassette to finish the test procedure. When applied to the sample well, the extracted specimen migrates across the test strip by capillary action. SARS-CoV-2 antigen, if present in the extract, binds to the antibody conjugates, and the immunocomplex is then captured on the membrane by the pre-coated anti-SARS-CoV-2 antibody, forming a colored Ag line that indicates a COVID-19 positive test result. For each patient, RDT was performed immediately after sample collection, and result reading was performed at 15 min after adding the extracted sample to the test cassette.The OnSiteRT-PCR testing was carried out within 24 h of sample collection by the Molecular Diagnostic Laboratory of LSU, an ISO 15189 and ISO 15190 accredited facility, and results were submitted to the national database. The test was performed targeting SARS-CoV-2 N1 and N2 genes, with human RNaseP as assay control target, after standard RNA extraction by MagMAX™ Viral/Pathogen II (MVP II) nucleic acid isolation kit using King Fisher Flex high-throughput automated extraction system . Primers recommended by Center for Disease Control (CDC) were used for the RT-PCR assay. In brief, we used Thermofisher TaqPath™ 1-Step RT-qPCR mastermix , no ROX. following a developed multiplex RT-PCR program on an ABI 7500 Fast DX instrument . 20 μL mastermix containing 5 μL of template was used for each test. The reaction condition includes steps of cDNA synthesis (10 min/55 °C), a hold step (1 min/95 °C), and subsequently 45 cycles of denaturation (10 s/95 °C) and annealing/elongation (30 s/55 °C) . The thrhttps://clades.nextstrain.org/ (accessed on 3 October 2021)) with default parameters [For SARS-CoV-2 circulating variant screening, we sequenced spike protein using specific primer sets from ARTIC nCoV-2019 V3 panel. Among the 133 RT-PCR positive samples, 69 samples with low Ct (<27.00) values were sequenced. Sanger sequencing was performed in an automated ABI 3500 XL genetic analyzer  described elsewhere . The rawrameters .The study was approved by the institutional ethical committee (ERC) of the International Center for Diarrhoeal Disease Research, Bangladesh . At the time of enrollment, written informed consent was obtained from each adult participant. For the children aged 2–10 years, consent was obtained from the parents or legal guardian and in the cases of children between 11–17 years in addition to written informed consent, verbal assent was also obtained.Data were computed in an Excel sheet. Statistical analysis was carried out using GraphPad Prism 8.0.2  and Stata version 15.1  were used for all analysis. Paired Student’s t-test was used to compare the mean and Wilcoxon rank-sum (Mann-Whitney) test was used to compare the non-normally distributed continuous data. To calculate sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and respective confidence intervals, diagti command was used. Receiver operating characteristics (ROC) curves were built using MedCalc Statistical Software version 20.013  for the analysis of sensitivity and the specificity of the RDT. A heat map was also generated to evaluate the relation among symptoms, days passed after the onset of the symptoms as well as the ability of the rapid test to detect COVID-19 at various time points.p = 0.26).A total of 380 individuals were enrolled where 230 (60.5%) were male with the median (IQR) age 33 (27–42) and 150 (39.5%) were female with the median age 32.5 (25–42). There was no significant difference between median age (p < 0.001)Out of 380 patients, 133 (35.0%) tested positives in RT-PCR where Ct values for N1 and N2 ranged from 11.0–31.7 (mean ± sd: 18.2 ± 4.7) and 11.0–36.1 (mean ± sd: 19.1 ± 5.0) respectively. The mean difference between the Ct values for N1 and N2 was significant  tested negative by the Onsite® COVID-19 Ag RDT. The kit’s sensitivity and specificity values in this study were therefore 91.0% (95% CI: 84.8–95.3%) and 99.2% (95% CI: 97.1–99.9) respectively. The positive and negative predictive values (PPV and NPV) are 98.4% (95% CI: 94.2–99.8%) and 95.3% (95% CI: 92.0–97.6%). When comparing the performance of the OnSite® COVID-19 Ag RDT with RT-PCR, it shows a very high concordance , as depicted in the ROC curve in Among the 133 study participants classified as positive by RT-PCR, 121 (91.0%) tested positive by the ® COVID-19 Ag RDT found at the cut-point 25.47 for N1 and 27.48 for N2 with sensitivity of 98.35%  variant (84.06%), 8 beta (South African) (11.59%) variant and 3 eta (20A/S:484K probably Nigerian) variant (4.35%). The RDT could detect all the samples that were sequenced indicating the capability to detect different variants of SARS-CoV-2. The summary has been depicted in the OnSite COVID-19 Ag rapid test kit as an easy-to-use test to detect COVID-19. The high sensitivity and specificity of the OnSite COVID-19 Ag rapid test obtained in this study, indicate a very robust performance of the test kit. The RT-PCR positive samples that have been missed by RDT, have mostly very high Ct values (>27) . The false positive samples were tested with an additional RT-PCR protocol and reconfirmed as a true negative. These samples were also negative for other COVID RT-PCR target i.e., SARS-CoV-2 virus genome, E-gene, N-gene and RdRp gene [Several rapid test devices have been evaluated so far in various platforms, and most of those were conducted either with archived samples, samples preserved in VTM or a limited sample size for prospective studies ,28,29,30es (>27) . Only twdRp gene ,32. TM COVID-19 Ag Rapid Test Device  and StandardTM Q COVID-19 Ag Test  found the overall sensitivity of these kits were 89% and 85.5%, respectively, and with specificities of 99.7% and 100% respectively [TM COVID-19 Ag Rapid Test Device found the sensitivity and specificity of 79.6% and 100%, respectively. It was found that RDT negative samples were not infectious although positive by RT-PCR. However, the number of the positives samples in this study was small (only 43 samples positive by both RT-PCR and rapid test with 11 discordant results out of 412) [TM COVID-19 Ag Rapid Test Device was also used in a prospective study with 634 asymptomatic patients in Spain, of whom 79 were positive, and the kit was able to detect in 48.1% cases only (38 patients) [®, Osong, Republic of Korea) was compared with RT-PCR of Allplex™ 2019-nCoV Assay  among 454 patients of which 60 people were positive and found 98.33% sensitivity. This high sensitivity might be explained by the small sample size in the study, and mainly by the low sensitivity (90%) of the RT-PCR kit, which will likely miss the low viral load samples, that would also be missed by the rapid test [A study evaluating the performance of Panbioectively . Another of 412) . The Panatients) . Howeverpid test . Howeverpid test .OnSite COVID-19 Ag rapid test kit in case of Ct values of 25.47 and 27.48 for N1 and N2 target respectively. Comparatively, AFIAS COVID-19 Ag test and ichromaTM COVID-19 Ag test  showed 100% sensitivity and specificity for Ct values <25.0 for RdRp and N gene. However, for E gene, the sensitivity falls to 91.3% and 95.7% respectively. For Ct values ranging from 25–30, the sensitivity reduces drastically to 34–64.4% [From the ROC curve it is evident that maximum sensitivity of 98.35% can be obtained with the 34–64.4% . In anotOnSite COVID-19 Ag Rapid Test kit showed high performance in determining positive patients that came to test after 1 to >6 days from the onset of symptoms. The heat map also indicates that patients of all age groups had come to the test facility as well as that the positive samples are well distributed among the age ranges. However, fever, cough, sore throat, and body ache seemed prominent symptoms among the COVD-19 positive patients. The result also portrays its capability to detect major circulating variants of concern (VOC) as well as variants being monitored (VBM).This OnSite COVID-19 Ag Rapid Test is a cost-effective easy to use test that does not require specialized equipment or skilled laboratory personnel. Besides, there is minimal chance of cross-contamination of samples, as the kit is designed for individual patients and limits hazardous features to the operator, as the virus is inactivated by the extraction buffer media. The high sensitivity and specificity of the kit also justifies its ability to be a viable alternative to RT-PCR.In the community and healthcare facilities, it often takes a significant amount of time from sample collection to RT-PCR test, from multiple hours to often even days. There is also a chance of cross-contamination of the samples that may render false-positive results when testing in batch form. One of the common pitfalls of RT-PCR is that it is also costly, and require skilled personnel along with sophisticated equipment, making it unavailable in resource-limited settings. In contrast, the The main limitation of the rapid tests is that there is possibility of false negative results in samples with low viral load. Hence, it might not be often suitable for detection of COVID-19 in less symptomatic and asymptomatic patients. OnSite COVID-19 Ag Rapid Test is a very reliable, fast, user-friendly and economic test, that can easily be implemented as the test of choice for rapid detection of COVID-19 in all sorts of healthcare facilities.Many countries worldwide are now shifting towards rapid Ag-based detection systems in the healthcare facilities for diagnosis of COVID-19, to reduce time as well as costs. However, RDT negative symptomatic samples should be confirmed by RT-PCR. It is likely that nasopharyngeal samples have slightly better sensitivity (92.48%) than nasal swabs only, but sample collection is less invasive to the patient when using nasal swabs. The sensitivity obtained for nasal samples should be comparable to self-testing performance at the community level, whereas nasopharyngeal sample could be an alternative for professional testing at the healthcare facilities with improved diagnostic capacity. Our study demonstrates that the


The study aimed to compare the Fassisig for 5 min at 20°C. The serum was analyzed using the FB-IgG according to the manufacturer’s specifications. Serum IgG concentrations were also determined by enzyme-linked immunosorbent assay (ELISA-IgG). FPT was defined as a serum IgG concentration <10 mg/mL.A total of 277 calves of 1-10 days of age were used in this study. Blood samples were obtained, and serum was extracted by centrifuging the samples at 2740× The mean ELISA-IgG serum concentration was 8.40 mg/mL . FPT prevalence based on the ELISA measurements was 66.8%. The prevalence of partial and full FPT based on the FB-IgG was 54.5%. The ELISA-IgG and FB-IgG results were subjected to correlation and regression analysis. Overall sensitivity and specificity of the FB-IgG were 61.1% and 58.7%, respectively. A statistically significant dependence on age was identified in the results.Our findings suggest that the FB-IgG rapid method is less accurate and provides no other advantages over established methods. Timely and adequate uptake of colostral immunoglobulins (Ig) by calves after birth is critical for disease prevention and later milk production in dairy cows ,2. If th® Bovine IgG (FB-IgG) , has been available for several years. No independent scientific review has yet compared the effectiveness of the FB-IgG and enzyme-linked immunosorbent assay (ELISA)-IgG assays at determining IgG concentrations. The only two currently available verifications are provided in the manufacturer’s insert /(1+ exp[–1.60+1.839˙lgELISA]). This equation models the probability of detecting an adequately supplied calf independent of the true ELISA-IgG concentration. The OR value revealed a 6.29-fold increase in the odds that a calf will be determined to be adequately supplied based on the FB-IgG if the ELISA-IgG concentration is greater by 1 decimal.The mean IgG concentration determined by ELISA-IgG was 8.40 mg/mL (range=0.10-47.50 mg/mL), and the distribution of the original values was skewed to the right. Among the 277 samples tested, the ELISA-IgG results indicated that 92 samples had an IgG concentration ≥10 mg/mL and 185 samples had an IgG concentration <10 mg/mL (indicating FPT). Thus, 66.8% of the calves examined had FPT according to the ELISA-IgG analysis. In comparison, the FB-IgG results indicated that 126 (45.5%) samples had a score of 1, 49 (17.7%) samples had a score of 2, and 102 (36.8%) samples had a score of 3. The FB-IgG results significantly correlated with the ELISA-IgG results (p<0.0001). However, Spearman’s rank correlation coefficient was low at rs=–0.36 . The equTo determine the sensitivity and specificity of the FB-IgG, the pFPT  and FPT  results (n=116) were once more combined into a single group and compared with the sufficiently supplied (score=1) values. The FB-IgG correctly identified inadequately supplied calves with an estimated sensitivity of 61.1% and an estimated specificity of 58.7% . We perfs=–0.36). In this study, FPT was defined as an IgG value of ≤10 mg/mL, which is consistent with the recommendation of the test manufacturer and published literature [The present study compared the accuracy of the FB-IgG and ELISA-IgG in assessing serum IgG levels to diagnose FPT in calves. Although the rapid method and the ELISA-IgG results were highly significantly correlated, the strength of the correlation was only moderate  of the calves assayed. When the age classes were evaluated separately, 72.0% of calves up to 3 days old and 49.7% between 4 and 10 days old were correctly classified. The data provided in the FB-IgG instructions appear to be inconsistent with the data obtained in the present study. Two studies are cited in the FB-IgG documentation: A 2014 study performed at the University of Goettingen and a 2014 study by “ID Ringbox IgG” . These tTherefore, we conclude that established indirect rapid methods, such as total serum protein determination or measurement of the serum gamma-glutamyl transferase activity, provide significantly more reliable results than the FB-IgG rapid method for detecting FPT in calves.MH: Performed animal experimentation, biochemical assays, analyzed and interpreted the results, and wrote the manuscript. SS: Organized the implementation of blood sampling on farms in Thuringia. KF: Performed the statistical analysis. AW: Designed the study, coordinated the research activity planning, and edited the manuscript. All authors read and approved the final manuscript.


Coronavirus disease 2019 is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is highly transmissible. Early and rapid testing is necessary to effectively prevent and control the outbreak. Detection of SARS-CoV-2 antibodies with lateral flow immunoassay can achieve this goal. In this study, SARS-CoV-2 nucleoprotein (NP) was expressed and purified. We used the selenium nanoparticle as the labeling probe coupled with the NP to prepare an antibody (IgM and IgG) detection kit. The detection limit, cross reaction, sensitivity and specificity of the kit is verified. Separate detection of IgM and IgG, such as in this assay, was performed in order to reduce mutual interference and improve the accuracy of the test results.The final purity of NP was 91.83%. Selenium nanoparticle and NP successfully combined with stable effect. The LOD of the kit was 20 ng/mL for anti-NP IgG and 60 ng/mL for anti-NP IgM, respectively. The kit does not cross reaction with RF. The sensitivity of the kit was 94.74% and the specificity was 96.23%. The assay kit does not require any special device for reading the results and the readout is a simple color change that can be evaluated with the naked eye. This kit is suitable for rapid and real-time detection of the SARS-CoV-2 antibody IgG and IgM. I.Beginning in December 2019, several unexplained pneumonia cases occurred in Wuhan City, Hubei Province. Chinese researchers identified the cause of the disease as a new type of coronavirus, namely severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) The LFIA is a fast, simple, accurate, stable, and portable diagnostic tool II.A.2SeO3) were obtained from Xiya Reagent . Bovine serum albumin (BSA) was purchased from Sigma. The anti-NP IgM and anti-NP IgG antibodies (Abs) were acquired from the Beijing Biodragon Immunotechnologies Co. Ltd . Deionized water (Millipore Milli-Q Grade) with a resistivity of 18.2 Polyethylene glycol (PEG) was purchased from the YuanYe Biological Technology Co., Ltd . Ascorbic acid (Vc) and selenous acid (HB.The pET28a(+)-nCoV-nucleoprotein-histidine tag plasmid was constructed, and the SARS-CoV-2-N-His recombinant protein was produced using E. coli BL21. The plasmid was transformed into E. coli BL21, and a single clone was inoculated into 5 mL of 2YT medium and cultured at 37°C and 220 rpm for 14 h. Subsequently, 2 mL of the bacterial solution was added into 1 L of 2YT medium and cultured at 37°C and 150 rpm for 2.5 h. The bacterial broth was incubated with 0.1 mM isopropyl C.2CO3. Then, recombinant nucleoprotein was added in drops to 100 mL SeNps with gentle stirring at room temperature. The mixture was then softly agitated for 30 min to allow conjugation of recombinant nucleoproteins to selenium nanoparticle surfaces by physical adsorption. Subsequently, 1 g bovine serum albumin (BSA) was added to the mixture for 30 min to block the non-coated selenium nanoparticle surfaces, and then the mixture was centrifuged at 10,000 rpm for 10 min. Selenium nanoparticle-conjugated recombinant nucleoprotein in a soft-pellet form was collected. Finally, the labeled proteins were resuspended in a working solution  and then stored at 4°C before use. The optimal pH was selected according to color development. At the selected optimal pH, 1.5 mg/mL, 2 mg/mL, and 4 mg/mL were selected as antibody coating concentration at the test line. The selenium nanoparticle solution (SeNps) was prepared using ascorbic acid to reduce selenite. The selenium nanoparticle preparation was used to label recombinant nucleoproteins at different pH levels, and the pH of SeNps was adjusted with KThe internal structure of the kit is shown in D.The anti-human IgM antibody or anti-human IgG antibody was sprayed on the test area as M line or G line by dispenser and sprayer (XYZ 3010), and the anti-His antibody was coated on the control line. After the coating was completed, the reaction pad was dried at 37°C for 4 h. The sample pad was immersed in the sample pad treatment solution  for 30 min and dried at 37°C for 4 h. The conjugate pad contained immobilized selenium nanoparticle recombinant nucleoprotein complex. The test strips were prepared prior to performing the assay, assembled, cut, and ready for use.E.We got commercially calibrated antibodies  diluted in negative serum as standards. Samples containing different concentration of anti-NP IgG (0–1000 ng/mL) anti-NP IgM (0–1000 ng/mL) in negative serum were applied on the sample pad for the limit of detection (LOD) assay in triplicate. The results could be determined by the naked eye within 10 min. At the same time, the test results were analysed by ImageJ and GraphPad Prism 8 to assist in determining the LOD. The detection of human IgG and IgM is easily affected by rheumatoid factors (RF), so it was necessary to test whether the kit has cross reaction with RF. Different concentration of RF (0–50U) were detected by this lateral flow kit.F.Fetal bovine serum (FBS) was chosen as the negative sample for the selection of kit conditions in the early stage. The serum of two healthy persons was used to verify the above conditions. SARS-CoV-2 positive specimen and negative specimen were used to validate the test strips. Among them, 19 cases were diagnosed as positive in Henan Provincial People’s Hospital, the COVID-19 designated hospital in Henan Province; 41 cases were confirmed negatives by clinical detection from the First Affiliated Hospital of Henan University, 12 normal persons who had no cold or fever symptoms within 2 weeks were also recruited into the study. For each kit, The sensitivity, specificity, positive predictive value, and negative predictive value of the kit were calculated according to the following formulas III.A.As shown in B.The conjugating concentration is the amount of nucleoprotein added to the selenium nanoparticle solution per unit volume. The coating concentration is the concentration of antibody sprayed on the test line. Nucleoprotein was labeled with selenium nanoparticles as follows. The normal conjugating concentration was  and 7.0 . Therefo and 7.0 . However and 7.0 . Based o and 7.0 , 3e, ind and 7.0 .Fig. 3.Using pH 10.0 as the conjugating condition, various conjugating concentrations and coating concentrations were tested. When the conjugating concentrations were The SARS-CoV-2 antibody screening kit requires high sensitivity. A clinical sample validation study was therefore performed, using a conjugating concentration of C.2 =0.982), respectively. The intensity of orange color was proportional to the anti-nucleoprotein IgG and anti-nucleoprotein IgM concentration in the range of 20–1000 ng/mL  anti-nucleoprotein IgM  in negative serum were tested by the corresponding kit in triplicate. The analysis process was completed in less than 10 min, and the LOD of anti-nucleoprotein IgG was 20 ng/mL , 5b with00 ng/mL  and 100–00 ng/mL . The kit00 ng/mL , 6b.FigD.A total of 53 samples were collected from the hospital, school and clinically diagnosed as negative for SARS-CoV-2. 39 patients and 12 normal persons were found to be IgG or IgM negative by testing and 2 were positive, including one IgG-positive sample and one IgM-positive sample . In addition, 18 cases were found to be positive using our kit , twelve IV.The structural proteins of SARS-CoV-2 include spike protein, envelope protein, membrane protein, and nucleoprotein. Spike protein infects host cells by binding to the human ACE2 receptor. This spike protein shows great variability, but the receptor is suitable for drug development; however, the sample detection accuracy is likely to decrease as the SARS-CoV-2 mutates. Nucleoprotein is the most highly conserved and strong immunogenic of these structural proteins, so the sample detection rate can be improved through use of nucleoprotein Antibodies will not be produced until about 10 days after infection, so the detection results by this kit of samples within 10 days of infection may be negative. Anti-spike protein antibody and anti-nucleoprotein antibody could be produced and secreted in the body after infected by SARS-CoV-2; and the nucleoprotein was selected to prepare the detection kit for antibody of SARS-CoV-2, therefore, this kit may not be able to detect samples that are antibody positive for spike protein.When the effect of coating concentration was tested, FBS was selected to ensure the stability of the detection matrix. The serum component of FBS is less than that in human serum component resulting in less interfering factors. The coating concentration of the test strip 2 mg/mL anti-human IgG antibodies and 4 mg/mL anti-human IgM antibodies did not have false positives. However, when human serum matrix was tested, it was found that the anti-human IgG antibodies coated with 4 mg/mL showed false positives. In addition, coating with 2 mg/mL and 4 mg/mL anti-human IgM antibodies both showed false positive results. Therefore, 1.5 mg/mL anti-human IgM antibody was used for coating in test line.The detectable antibodies of SARS-CoV-2 include IgG and IgM, which can be completed using two kits separately or using one kit which including IgG and IgM test line. The detection speed and method of interpreting the kit results are the same. In the separate detection strategy, there is no mutual interference between the target IgM and IgG, and the accuracy is higher compared with the combined detection strategy. A combined test can reduce the sample volume but the process requirements are higher than those of using two separate tests. In order to quickly develop a SARS-CoV-2 antibody detection kit, we here selected the separate test method, and we planned to develop a test kit for combined detection. Many antibody detection studies for SARS-CoV-2 based on colloidal gold were reported, and the results were shown in In this study, only preliminary testing of clinical samples was performed. A total of 19 positive samples were tested, and 18 cases were consistent with nucleic acid results. Of the 19 patients, 9 were male and 11 were female. They were all severe and critical condition patients. One patient was 20–30 years old, three patients were 40–50 years old, four patients were 50–60 years old, and 11 patients were over 60 years old. The older patients might have compromised immunity, making them prone to be infected. Seven patients had diabetes, five patients had coronary heart diseases, and four patients had high blood pressure. Four of them had both diabetes and coronary heart disease, suggesting that patients with these diseases may be more susceptible to COVID-19. The patient who had the negative result had chronic bronchitis. Whether chronic bronchitis affects the detection of SARS-CoV-2 requires further research. To determine the true effectiveness of the kit, it is still necessary to expand the test sample size to validate the overall sensitivity and specificity of the kit. Moreover, patients having different stages of COVID-19 and a wide range of ages need to be tested to determine whether the detection results of the kit are affected by those factors.V.We have developed a new assay for the rapid detection of SARS-CoV-2 antibody (IgM + IgG) using a selenium nanoparticle based method. The LOD was 20 ng/mL for anti-NP IgG and 100 ng/mL for anti-NP IgM, respectively. The sensitivity of the kit is 94.74% and the specificity is 96.23%. The assay is portable and fast. The results can be obtained within 10 min. The assay kit does not require any special device for reading the results and the readout is a simple color change that can be evaluated with the naked eye. This kit is suitable for rapid and real-time detection of the SARS-CoV-2 antibody.


The variant 20I/501Y.V1, associated to a higher risk of transmissibility, emerged in Nice city  during January 2021. The pandemic has resumed late December 2020 in this area. A high incidence rate together with a fast turn-over of the main circulating variants, provided us the opportunity to analyze modifications in clinical profile and outcome traits. We performed an observational study in the University hospital of Nice from December 2020 to February 2021. We analyzed data of sequencing of SARS-CoV-2 from the sewage collector and PCR screening from all positive samples at the hospital. Then, we described the characteristics of all COVID-19 patients admitted in the emergency department (ED) (n = 1247) and those hospitalized in the infectious diseases ward or ICU (n = 232). The UK-variant was absent in this area in December, then increasingly spread in January representing 59% of the PCR screening performed mid-February. The rate of patients over 65 years admitted to the ED decreased from 63 to 50% (p = 0.001). The mean age of hospitalized patients in the infectious diseases ward decreased from 70.7 to 59.2 (p < 0.001) while the proportion of patients without comorbidity increased from 16 to 42% (p = 0.007). Spread of the UK-variant in the Southeast of France affects younger and healthier patients. A matched cohort study including 109,812 patients in the UK showed an increase in deaths from 2.5 to 4.1 per 1000 cases detected associated to the 20I/501Y.V1 variant2.Starting in September 2020, the new SARS-CoV-2 variants called VOC-202012/1  have rapidly spread in the UK, associated to a higher risk of transmissibility3. It was reported in France late December 20205.The spread of this variant (20I/501Y.V1 i.e. “UK-variant”) in continental Europe was identified late December 2020, as reported by the European Center for Disease Prevention and Control (ECDC) with the first reports in Netherland, Belgium, Denmark5.After a second outbreak in November , the pandemic declined in France in December 2020. On December 10th, the administrative area around Nice, French Riviera, in the Southeast of France, which totalizes more than 1 million inhabitants, exhibited a stable incidence of new positive cases at 169/100,000, i.e., a slightly higher value relative to the whole French territory. Contrary to the rest of France, the pandemic has resumed in this area since late December, reaching an incidence of 583/100,000 on February 13thAt the same time, the UK-variant, emerged in January in our area. The specific epidemiologic trends of COVID-19 epidemic in our area, i.e., a high incidence rate together with a fast turn-over of the main circulating variants, provided us the opportunity to analyze the UK-variant-related clinical profile and outcome traits.The survey in sewage of the city of Nice showed that the UK-variant represented 2.6%, 8.3% and 79.1% of all identified SARS-CoV2 strains in December 2020, January 2021, and February 2021 respectively.Consistent with these data, the random sequencing of positive PCR recorded from December 18th to 26th at Nice University hospital showed no identification of the UK-variant. This analysis was coordinated by the French National Center for Coronavirus  and was performed on 25% all the 73 positive patient’s samples isolated during this period.Finally, a systematic screening PCR to identified latent variants was scheduled starting late January, on governmental recommendation. At this time, all positive PCR done at the Nice Hospital were screened. The rate of UK-variant during Aft1-F was 20% of all positive PCR, then 59% during Aft2-F.Altogether, these data suggested that the UK-variant was absent in our area in December 2020, then increasingly spread in January 2021.From December 1st to February 28th, 1247 patients were admitted for a suspected or diagnosed SARS-CoV-2 infection (Elderly Care Facilities people not included), aged 66.1 ± 17.9 [17–102] years, 54.0% male gender. Six patients (0.5%) died in the ED, while n = 731 patients (58.6%) were hospitalized in a medical ward and n = 109 (8.7%) in ICU; the remaining, n = 401 (32.1%) were sent back home (ambulatory follow-up). Patients and clinical characteristics are reported  and hospital location after ED  over these 3-month period are reported Fig. .Figure 1Until February 22nd, we identified 29 hospitalized patients bearing the UK-variant. After pairing cases (n = 29) versus controls (n = 58) on age (61.0 ± 10.7 vs 61.6 ± 11.9 years) and gender , we compared time 1st symptoms-ED admission and the ED NEWS-2 score (cases vs controls) respectively: 7.3 ± 3.5 vs 6.8 ± 3.9, p = 0.424 and 6.2 ± 2.4 vs 6.4 ± 3.0, p = 0.642. Patients admitted in the ED required immediate ICU management in respectively 10.3% (3/29) and 12.0% (7/58), p = 0.810.The three a posteriori defined groups, i.e., Bef-F, Aft1-F and Aft2-F respectively, included 51, 72 and 54 patients. Comparison of main data between groups are reported .In patients under 65 years old hospitalized in ID department (n = 76), the rate of patients without any comorbidity rose from 21% (Bef-F) to 50% (Aft2-F), p = 0.029.4 and therefore the risk of an increased number of patients requiring an ICU bed. Beyond the good organization of the flow of an increased number of patients, the question arises as to whether this lineage is also associated with a more severe clinical presentation, which could involve younger patients. Our results show that patients admitted in January and February 2021 in ED for a COVID-19 and therefore hospitalized for a COVID-19-related pneumonia are significantly younger with around 50% of them aged less than 65 years old. Our data strongly support the increased risk of SARS-CoV-2-related severe pneumonia in younger patients contemporaneously to the spread of the UK-variant in our area.The occurrence of UK-variant at the end of 2020 is of major concern, because of the contagiousness issueThe severity issue is less conclusive. The rate of ED patients admitted in ICU increases, but an increased clinical severity is not obvious, as neither the NEWS-2 score nor the main signs of clinical severity  have changed over time. Even when UK-variant patients were paired on age and gender, no difference in term of early-stage severity was evidenced. In the same way, patients hospitalized in the ID department did not show a more severe inflammatory response or extended CT scan lung injuries at later times, i.e., after the spread of the UK-variant. Finally, for ICU patients, neither the severity score at admission (SAPSII) nor the depth of the respiratory distress seemed to increase by the variant. Altogether, there is no evidence to support a different and/or more severe clinical picture of the SARS-CoV-2-related pneumonia with the UK-variant.7. Importantly, we provide herein data in favor of an increased ratio of healthy patients  hospitalized for a SARS-CoV-2 related pneumonia associated to the spread of the UK-variant in the fifth-largest city of France. Combined to the patient’s younger age, this brings several concerns. The most immediate is about the risk of having a pandemic evolving toward young and healthy populations, with even greater social consequences. Another consequence, supported by our data is the increase of patients with younger age/comorbidity free-related ICU requirement. So far, older patients with comorbidities have a greater risk of ICU admission9. The increase of younger people admitted in ED for a COVID-19, in our cohort, is associated with a trend of increased ICU ratio requirement afterward. The same trend was noticed in the ID department. As the severity is not obviously increased, a likely explanation is that physicians, after months of fighting against the pandemic with critical patients mainly over 65 years old, have been surprised to receive younger patients with severe pneumonia, leading possibly to a higher ICU demand. As the pandemic rises concerns about ICU being overwhelmed, such change in patient’s profile brings new insights in the debate about ICU triage11. Furthermore, according to the country of interest, the vaccination strategy may have to be reconsidered in order to include a younger part of the population at risk of severe or critical COVID-19. Finally, the effect of the UK-variant on SARS-Cov-2-related mortality cannot be appropriately assessed here because, giving a mortality rate under 0.5%, the required sample size needs to be thousands of patients2. The number of patients managed in our hospital and the duration of follow-up available do not allowed us to consider the final outcome for comparison between study periods.The occurrence and severity of COVID-19 has been related to comorbidities like diabetes, hypertension or obesity12 and may have play a role in the very low case fatality rate in this country13. Spike mutation D614G that probably occurs in China before its diffusion in Europe is associated with a decreased age of COVID-19 patients possibly due to an increased viral load in younger patients14. The N501Y mutation is associated with adaptation to rodent, for instance mice15 and may increase SARS-CoV-2 Spike protein binding to ACE2 because of conformational changes, thus increasing its transmissibility1. The role of ∆69/∆70 on the spike protein is also potentially involved in the increased transmissibility16.Until today, reports of a relationship between SARS-CoV-2 genetic specificities among variants and clinical presentation are scarce. One variant with a deletion (∆382) in the open reading frame 8 have been associated to milder infections in Singapore4. Since our data are mainly on hospitalized patients, the carriage of the UK-variant in a younger population bears further studies about the clinical fraction associated to the variant in the population17 outside the hospital. A puzzling question is why younger patients need now to be hospitalized, which was not the case before? The higher transmissibility of UK-variant is supposed to be similar among all age groups18. As of 26th of February, 60,000 inhabitants of Alpes Maritimes had received two doses of vaccine. Increased herd immunity after vaccination of the old community and mortality during the two previous epidemic peaks in France that has affected the most fragile elderly may have independently contributed to the observation of fewer patients over 75-year-old requiring hospitalization.The spread of the UK-variant was found associated with a higher transmissibility as reported by others2 and our study wasn’t designed on purpose.Our study has some limitations. First, data are retrospective. Then, data on the UK-variant spread results from analysis of sewage or selected hospital samples and not from a systematic survey. Data on hospitalized patients (variant versus “common” strain) are incomplete. Finally, patient’s data were extracted from ED, ID, and a medical ICU of the University hospital of Nice, but patients hospitalized in other hospitals from the ED were not included in the study, leading to incomplete analysis of involved patients. Our study does not allow to distinguish an effect of the UK-variant at the individual level, that is a higher probability to develop pneumonia after a contact with the virus due to the Spike protein RBD mutations, from a modification of virus diffusion in the Southeast of France population leading to a higher exposure of younger inhabitants which could be related to the virus itself or to a modification in non-pharmaceutical interventions adherence. Finally, the effect of the UK-variant on SARS-Cov-2-related mortality cannot be assessed here because, giving a mortality rate under 0.5%, the required sample size needs to be thousands of patientsIn summary, our data alerts on the consequences of the UK-variant spreading with younger and healthier patients requiring hospitalization for a SARS-CoV-2 pneumonia. Health system impact  as well as social consequences should be assessed in larger prospective studies. The spread of SARS-CoV-2 variants displaying a higher transmissibility may be associated with an increased diffusion across younger ages groups. Therefore, vaccination strategies and preventive nonpharmaceutical interventions need to integrate people less than 65 years-old even those without any medical history.We designed an observational study to assess the clinical profile and outcome of patients infected with the SARS-CoV-2, before and during the spread of the UK-variant. Patients admitted in the Emergency department (ED), in the Infectious Diseases department (IDD) and the ICU at Nice University Hospital (Cote d’Azur University UCA), France, were part of this study. Data were extracted from the hospital electronic database (n°410) of patient’s record, registered on clinicaltrial.gov NCT04779021, also from the hospital Virology Lab and the Institut de Pharmacologie Moléculaire et Cellulaire, UMR7275 CNRS/UNS .In the way to quickly characterize the clinical consequences of the spread of these new variants, we designed a three-step approach.First step: Spread of the UK-variant in our area and among hospitalized patients. Data of the sequencing of SARS-CoV-2 identified from the sewage collector of the city of Nice  in December and January, and PCR screening from all positive samples analyzed at the hospital Virology Lab from newly admitted and diagnosed patients (starting late January 2021).Second step: Concomitant epidemiology and clinical characteristics at hospital admission of COVID-19 overtime, by an evaluation over a three-month period, from December 1st, 2020, to February 22nd, 2021, of all patients admitted in the ED for COVID-19. Demographic data, clinical severity as recorded by the NEWS-2 scoring system and the follow-up (ward admission versus ambulatory follow-up). Patients coming from Elderly Care facilities clusters or transferred from another hospital with a SARS-CoV-2 positive PCR were not included in the study.Third step: Clinical severity and follow-up of hospitalized patients for a SARS-CoV-2 pneumonia, in the IDD or ICU over three fortnights:Bef-F),before the spread of the UK-variant in our area, i.e., from December 7th to 21st 2020 (Aft1-F and Aft2-F).the two fortnights after starting a PCR screening from all positive samples analyzed at the hospital Virology Lab from newly admitted and diagnosed patients (late January 2021), i.e., from January 24th to February 7th and from February 8th to 22nd, 2021 respectively Demographic data, clinical signs and severity were recorded by the NEWS-2 (ED), SAPS-2 and SOFA scores (ICU), CT-Scan findings and the follow-up were recorded and analyzed.https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye), data being analyzed with their pipeline (https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.html). For each position associated with 20I/501Y.V1 lineage  the fraction of 20I/501Y.V1 lineage reads relative to the total number of reads is assessed.We assessed the UK-variant spread in sewage through the Nice wastewater plant, which treats the sewage for a population of 390,000 inhabitants. The daily incoming volume varied from 97,000 m3  to 128,000 , due to rain at the end of January. Sequencing followed the protocol of the Artic consortium  or ViroBOAR Spike 1.0 RT-PCR  kits, following manufacturer’s instructions. Identification of the 20I/501Y.V1 variant was further verified by sequencing by sequencing a random set of samples in which the reliability of the RT-PCR screening tests was checked. Briefly, Spike protein regions covering ∆69/70 and ∆144 deletions, as well as the N501Y substitution, were amplified through RT-PCR, and amplicons were sequenced by the Sanger method.Patients who were hospitalized with an identification of the UK-variant at the Virology Lab (see upper) were paired (1:2) with patients hospitalized during the three first weeks of December (variant-free period), on age and gender. News-2 score, time 1st symptoms-ED admission and further hospital orientation  were the main evaluated criteria.®.Data were expressed as mean ± standard deviation SD. Comparisons are done using Chi-2, Fisher exact-test for categorical variables and Student-t test, Kruskal–Wallis test and Mann–Whitney U-test for continuous variables comparisons, if required. All data were analyzed using SPSS software


Trypanosoma cruzi, the causal agent of the Chagas disease, a NICT-like protein (Tc/NICT) was identified with a short C-terminus orthologous to the human protein, a large ectodomain (ECD) with numerous glycosylation sites and a single-core transmembrane domain containing a putative TM-domain (457GSVGA461) important for the γ-secretase complex activity. Results: Using the Spot-synthesis strategy with Chagasic patient sera, five extracellular epitopes were identified and synthetic forms were used to generate rabbit anti-Tc/NICT polyclonal serum that recognized a ~72-kDa molecule in immunoblots of T. cruzi epimastigote extracts. Confocal microscopy suggests that Tc/NICT is localized in the flagellar pocket, which is consistent with data from our previous studies with a T. cruzi presenilin-like protein. Phylogenetically, Tc/NICT was localized within a subgroup with the T. rangeli protein that is clearly detached from the other Trypanosomatidae, such as T. brucei. These results, together with a comparative analysis of the selected peptide sequence regions between the T. cruzi and mammalian proteins, suggest a divergence from the human NICT that might be relevant to Chagas disease pathology. As a whole, our data show that a NICT-like protein is expressed in the infective and replicative stages of T. cruzi and may be considered further evidence for a γ-secretase complex in trypanosomatids.Nicastrin (NICT) is a transmembrane protein physically associated with the polytypical aspartyl protease presenilin that plays a vital role in the correct localization and stabilization of presenilin to the membrane-bound γ-secretase complex. This complex is involved in the regulation of a wide range of cellular events, including cell signaling and the regulation of endocytosed membrane proteins for their trafficking and protein processing. Methods: In  Nicastrin (NICT) is a single-pass transmembrane glycoprotein that is part of the γ-secretase complex (GSC). This complex is present in all eukaryotic cells and functions in the maturation and processing of all type 1 proteins destined for cell membranes. In addition to NICT, the GSC contains three other compounds: presenilin (PS), anterior pharynx-defective 1 (Aph1) protein and presenilin enhancer 2 (PEN-2). Human nicastrin (hu-NICT) is physically associated with PS, with an important role in the stabilization and correct localization of PS to the membrane-bound GSC [Presenilin is a unique protease in that it cleaves within the lipid bilayer targeting many type 1, single transmembrane-spanning proteins that vary widely in their sequence and size ,3. NICT,The glycosylation of NICT appears to be critical for the substrate preference and enzymatic activity of γ-secretase . As a glTrypanosoma cruzi based on our characterization of a TM aspartyl protease that shows hallmarks of human PS [T. cruzi is the causative agent for American trypanosomiasis, more commonly referred to as Chagas disease, and infections are typically spread by triatomine vectors that affect millions of people throughout Latin America [T. cruzi NICT and developed antibodies against synthetic peptide sequences to identify the cellular localization of the protein. The immunological importance of one of these epitopes was examined by parasite adhesion inhibition study and as a possible immunological target. Other structural studies and database searches revealed that the Tc/NICT contains a large, heavily glycosylated extracellular ectodomain with five linear B-cell epitopes and expresses an important putative TM-domain for the γ-secretase complex activity. Phylogenetic analysis showed that the Tc/NICT is localized in an own subgroup with the T. rangeli. In the search for new drug targets in T. cruzi, the GSC is a promising candidate.While much is known about the GSC compounds in superior eukaryotic organisms, very little is known about their role in inferior eukaryotic cells. Recently, we showed the potential for the presence of a GSC in human PS . T. cruz America . In the N,N-dimethylformamide, Freund’s incomplete adjuvant, DAPI, TRITC and FITC labeled anti-rabbit IgG antibodies, TRITC-phalloidin, monodancylcadaverine, maleimide activated kit, Tween® 20, acetonitrile, monodancylcadaverine, tissue protease inhibitor cocktail and trifluoracetic acid were obtained from Sigma–Merck . Rabbit and goat alkaline phosphatase-labeled anti-human-IgG (AP-anti-huIgG) and anti-rabbit IgG (AP-anti-rabIgG) were purchased from Abcam . Super Signal R West Pico chemiluminescent substrate was from Pierce Biotechnology . Centrifugal Filter Units (cut-off 10 kDa) were from Millipore  and Nitro-Block II from Applied Biosystems, . Fetal bovine serum (FBS) was from Thermo Fisher Scientific Inc.  The brain and heart infusion (BHI) mediums were from Difco and nitrocellulose membrane from BioRad, .Amino-PEG500-UC540 cellulose membranes were obtained from Intavis AG Bioanalytical Instruments . Amino acids for peptide synthesis were purchased from Calbiochem-Merck . BSA, acetic anhydride, T. cruzi was obtained from Dr. Maria A. de Souza  and the Trypanosomatidae collection (CT–IOC/05). Parasites were maintained in BHI medium supplemented with 10% FBS [T. cruzi-infected Vero cell cultures, maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), at 4th day post-infection (dpi) [The CL-Brener strain of  10% FBS . Experimon (dpi) .T. cruzi, was represented by a continuous series of 144, 14-residue peptides that overlapped preceding and following peptides by 9 residues. The peptides were synthesized in place on a cellulose membrane according to a SPOT synthesis protocol [v/v) in N,N-dimethylformamide to render peptides unreactive during the subsequent steps. After acetylation, Fmoc protective groups were removed by the addition of piperidine to render nascent peptides reactive. The remaining amino acids were added by this same process of coupling, blocking and deprotection until the desired peptide was generated. After the addition of the last amino acid in the peptide, the amino acid side chains were deprotected using a solution of dichloromethane–trifluoracetic acid–triisobutylsilane  and washed with methanol. Membranes containing the synthetic peptides were either probed immediately or stored at −20 °C until needed. Negative and positive controls were included in each assay.The entire sequence (728aa) of the non-characterized protein nicastrin, deposited in the UNIPROT (Q4DEM3) and a possible component of GSC in protocol  using an® 20, pH 7.0) at room temperature under agitation or overnight at 4 °C. After extensive washing with TBS-T , membranes were incubated for 2 h with a pool of human patient serum (n = 7) diluted 1:250 in TBS-MT and then washed again with TBS-T. After that, membranes were incubated with rabbit alkaline phosphatase labeled anti-human IgG (diluted 1:5000), prepared in TBS-CT for 1 h, and then washed with TBS-T and CBS . The chemiluminescent substrate Nitro-Block II was added to complete the reaction.SPOT membranes were washed with TBS  and then blocked with TBS-MT  as described previously . BrieflyT. cruzi peptides, (Tc/NCT-1 = SLQDIIRGLSIPDT and Tc/NCT-4 = KSLRIPHGDW GATW), were chosen to be synthesized by the F-moc strategy in a synthesizer machine  with a C-terminal cysteine that was used to conjugate peptides to bovine serum albumin (BSA) using a maleimide activated kit according to manufacturer’s instructions. The reaction mixture was passed through a centricon-P10, and the peptide concentration in the filtrate (uncoupled peptide) was measured on a Qubit device . After the coupling reaction, two New Zealand rabbits were immunized by subcutaneous injection of peptide-BSA (150 µg) emulsified with an equal volume of saponine. Three other inoculations, without adjuvant, were each administered 7 days later, and the serum was collected five days after the last injection. Blood was collected under standard bioethics conditions from the marginal ear vein.Two Anti-Tc/NICT serology was performed on ELISA microplates  as previously described . In all T. cruzi epimastigotes (1 × 108) in the log phase that was collected by centrifugation (5000× g for 30 min at 4 °C) and washed three times in PBS (pH 7.2). Parasites in the pellet were suspended in extraction buffer  with 1% Triton X-100 and protease inhibitors) and subjected to six cycles of freeze-thawing. The detergent soluble fraction in the supernatant was collected after centrifugation . An extract of hydrophobic membrane proteins was obtained using a previously described method with adaptations [8) were collected, washed and lysed as described above. Before centrifugation, lysis buffer was added, and samples were kept on ice (4 °C) for 30 min with vortexing every 10 s. The suspension was centrifuged  to pellet cell debris, and the supernatant was incubated at 10 min at 37 °C to generate a phase separation by centrifugation . The aqueous phase with hydrophilic proteins was carefully removed, and the detergent-rich phase was processed for phase separation two more times. Protein was precipitated from the final phase by the addition of 4 volumes of cold acetone (−20 °C) followed by vortexing and a 1 h incubation at −20 °C. The precipitate was pelleted by centrifugation , and the pellets were air-dried before being solubilized in SDS-PAGE by a 5 min incubation at 100 °C. Protein concentration was measured using the Folin–Lowry method.Detergent soluble fractions were prepared from ptations . Brieflyw/v) SDS and 20% (v/v) methanol. The nitrocellulose was saturated with 5% (w/v) non-fat powdered milk in TBS-T, washed three times with TBS-T, and incubated overnight with 1:200 dilution rabbit anti-Tc/NICT-1 sera. The sheets were washed three times with TBS-T, and immunoreactive proteins were localized using HRP-anti-rabIgG (1:5000) for 1 h at 25 °C. Proteins were detected by chemiluminescence using the SuperSignal West Pico substrate kit . Revealed bands were quantified by densitometry using Image J .Protein samples were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gels electrophoresis  under reducing conditions . For WesT. cruzi epimastigotes extracts (detergent and soluble fractions) were obtained as described above and resolved by SDS-PAGE (10% under reducing conditions). After transfer to a nitrocellulose membrane, was used Immun-Blot® Kit for glycoprotein detection  following the manufacture’s instructions.g for 30 min at 4 °C), washed twice with PBS, fixed with paraformaldehyde (4%) in suspension for 5 min and washed 3× in PBS. The parasites were placed onto a glass slide and allowed to air-dry overnight. Next, cells were blocked with 1% casein in PBS (pH 7.4) for 15 min at 37 °C. Afterward, parasites were incubated with rabbit anti-Tc/NICT-1 sera (1:100) for 2 h at 37 °C. Slides were washed in PBS and then incubated with TRITC-labeled anti-rabIgG (1:400) for 1 h at 37 °C. Additionally, anti-β-tubulin (1:400) was used to probe parasites’ microtubules. Following this, parasites were washed in PBS, stained with DAPI and mounted in DABCO solution. An Axio Imager M2 fluorescence microscope (Carl Zeiss) was used to collect images.Parasites in the two different life stages, epimastigotes and trypomastigotes, were collected by centrifugation (5000× 4) were maintained in supplemented RPMI 1640 medium (10% FBS and 2% l-glutamine) at 37 °C in a 5% CO2 atmosphere and seeded in 24-well culture plates containing 13 mm2 glass coverslips. For the analysis of parasite-host cell interactions, culture-derived trypomastigote forms (106) of T. cruzi (CL strain) were incubated at 4 °C for 1 h with anti-Tc/NICT-1 sera  or pre-immune sera (1:50) diluted into RPMI 1640 medium with 0.5% BSA. Next, parasites were added to plated Vero cells at 37 °C. After 2 h, cultures were washed three times with PBS to remove unbound parasites and fixed in Bouin’s solution, followed by staining with Giemsa. The attached parasites were counted from 100 cells in random fields of view. Counts were compared to the untreated group (Control). Vero cells (5 × 10T. cruzi NICT sequence (Q4DEM3) was prospected in Blast (Delta-Blast) with a nucleotide database restricted Kinetoplast taxon. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [The ed model . The inied model ,25. T. cruzi NICT sequence (Q4DEM3) was performed using the human homologies to sequences already identified and annotated proteins in other organisms in the database UniProt  and nucleotide database restricted Kinetoplast taxon. The alignment of the sequences was performed using the T-Coffee server .The data bank search for http://web.expasy.org/protscale/, accessed on 15 January 2021) and TMHMM Server v. 2.0 . The prediction of the secondary structure of the protein was performed by the PSIPRED servers  and CDM  and the tertiary structure prediction and the transmembrane domain (TM) by the I-TASSER server . Some physicochemical parameters related to the peptides used in this study  were calculated with the ProtParam tool . The protein transmembrane orientation was obtained by inserting protein structure in the orientation of membrane protein database ) using the PPM server [http://lbqp.unb.br/NetWheels/, accessed on 10 August 2020). Moreover, the identification of nicastrin conserved motifs was performed on the MEME Suite server [www.uniprot.org, accessed on 15 January 2021).The hydropathicity scale of the protein was obtained with the ProtScale tool . The N-GlyDE web server is available at http://bioapp.iis.sinica.edu.tw/N-GlyDE/, (accessed on 15 November 2020).The predictions of glycosylation sites were realized using the NetNGlyc 1.0 Server  prior to the start of the study. The animals were housed and maintained according to the institutional guidelines for animal studies, which conform to the specifications set forth in the US National Institutes of Health guidelines for the care and use of laboratory animals. All efforts were made to minimize suffering.T. cruzi (n = 92) were obtained from the Parasitic Disease Laboratory, Oswaldo Cruz Institute/FIOCRUZ (CAAE: 52892216.8.0000.5248 and 1.896.362). The diagnosis as positive for infections by T. cruzi was confirmed before inclusion in the study both clinically through the clinical signs and symptoms as well as serologically through immunofluorescence and enzyme-linked immunoassay (ELISA). For this study, patient sera remained anonymous and were pooled for experiments.Sera from patients chronically infected with t-test was considered if p-value ≤ 0.05.Statistical analysis was performed using GraphPad Prism version 5.0. The statistical difference using a Epitopes in the putative Tc/NICT protein (728 aa) were identified based on recognition of peptides in a synthesized library (144 peptides) by Chagasic patient sera . The pattern of reactivity for the antibodies generated in infected patients demonstrated that a significate number of peptides were recognized A. An anaWhen comparing the characteristics of the epitopes, two peptides, epitopes Tc/NICT-1 and Tc/NICT-4 were determined to display the most intense antigenic determinants and chosen for generating polyclonal sera. For immunizing rabbits, the peptides were synthesized with one cysteine at the carboxyl-terminal (LQLDIIRCLSIPDTC and KSLRIPHGDWGATWC) to facilitate covalent conjugation to BSA. Two rabbits were independently immunized for each peptide, and serum was monitored weakly. After the fifth  immunizations, the titer against Tc/NICT-1 increased ~3.3 times (dilution 1:40/1:80) in relation to the Tc/NICT-4 . The immT. cruzi NICT protein. On Western blots, antibodies against the Tc/NICT-1 peptide detected in total extract only a single protein with a molecular mass of ~72 kDa  parasites C. A grapro cells D.T. cruzi, six sequences deposited in the NCBI data bank were aligned to compare the epitopes. This analysis showed that, in totality, all of the epitopes identified by sera from patients were identical to the different strain sequences of T. cruzi The high conservation of the structure of the epitopes suggests that a strong cross-immunity would be induced by immunization.To investigate the cross-immunity conferred by the Tc/NICT protein across the lineages of T. cruzi NICT-like protein. This predicted structural model displayed a high probability of a TM domain at amino acids 704–716 , in T. rangeli by GGVGS (aa 389–394), in T. brucei (aa 384–389) and T. congolense (aa 385–390) by GSIGS. In Arabidopsis thaliana by GYLGS (aa 402–407), in Chlamydomonas by GYMGS (aa 305–400), and in Dictyostelium by GYVGS (aa 300–305). Data available from studies of GSC in animals indicate that certain amino acid motifs are crucial for proteolytic activity, substrate recognition, and complex assembly . To veriThe other domains and properties analyzed compared to human nicastrin are shown in N-linked glycosylation is one of the predominant post-translational modifications involved in a number of biological functions. Several predictors were made available, and most of them evaluate their performance at every asparagine in protein sequences but are not confined to asparagine in the N-X-S/T sequence. Using this approach, 7 N-glycosylation sites 84 (NNS), 329 (NET), 425 (NICT), 436 (NFT), 526 (NTT), 580 (NRS) and 675 (NDS) were identified in the T. cruzi NICT-like protein  search criteria name: “nicastrin” and length: 300 TO 800). Information of the sequence Q4DEM3 in Uniprot server shows “Uncharacterized protein” in protein name, and protein status is predicted with domains showing to be nicastrin. Prospection in Blast with Q4DEM3 sequence restricted in Kinetoplastidae taxon returned only the Trypanosome species. The tree with the highest log likelihood is shown in A phylogenetic tree of NICT was retrieved on the Uniprot server , similar to other orthologous proteins. This protein possesses 18 cysteine amino acids that may indicate a complex extracellular structure dependent on SH bridges. Interestingly, none of the 18 Cys amino acids were located within the identified epitope structures.To characterize the putative  defined , confirmThe model also suggested that each epitope consisted of random coils and turns with no definable secondary structures. Based on a physical-chemical analysis of stability, net charge at pH 7.0 and hydrophobicity, which contribute positively to antibody production, two epitopes (Tc/NICT-1 and Tc/NICT-4) were chosen to be synthesized as four-component MAP for antiserum production. In addition, each peptide showed high reactivity against Chagasic patient sera by peptide-ELISAs . The choT. cruzi was subjected to phase separation using Triton-X114. Western blot analysis of hydrophobic proteins revealed a band at ~92 kDa suggesting the final mature form of Tc/NICT is highly glycosylated. This is consistent with the prediction for the presence of 7 N-glycosylation sites Tc-NICT protein has sites and explains the observations by immunoblot and molecular mass determination . The change in amino acids between I, L, M, and V should not affect the hydrophobic character of the domain. However, the 457D to G substitution could have a profound influence on the polar characteristics of this domain in Tc/NICT. Nevertheless, experiments performed in Dictyostelium [The phylogenetic analysis showed that the t clades , and theostelium , which hT. cruzi, the TM (aa 704–716) is localized more distal than the TM helix of the hu-NICT (aa 667–690), but both contain a hydrophilic patch that is important for TM–TM interactions. The short C-terminus appeared not to be structured, which was also see in the Tc/NICT similarly found for others NICT proteins [Another important structural residue implicated in the γ-secretase activity of human cells  is Glu-3proteins . In the proteins ,51. In cA major difference observed between hu/NICT and Tc/NICT was related to the number of predicted glycosylation sites present in the ectodomain (EDC). While the human presents 16 sites  and adopts a thermostable structure , the Tc/T. cruzi that includes its cellular localization and modeling. While the function of the T. cruzi NICT-like enzyme is not yet defined, our results show that it is expressed, and its subcellular localization is suggestive for a function in secretion and/or endocytic trafficking. The identification of NICT as a bona fide protein further supports the possibility of the formation of GSC in parasites. Antibody blocking studies suggest that it could play a role in parasite–host cell interactions. An intensive investigation of the spatial and temporal expression patterns of the T. cruzi NICT-like gene combined with the use of new knockout techniques should help reveal their relevant function in the host–parasite relationship.This is the first description of a NICT orthologue protein in T. cruzi expresses a nicastrin protein with structural properties similar to its orthologous human protein. The protein is predicted to have a single transmembrane domain with a long extracellular N-terminus that was corroborated by localization studies. Five IgG-binding epitopes were restricted to unfolded regions in the extracellular segment according to the structural model. Antibodies against Tc/NICT do not interfere significantly with the growth and viability of the parasite but can interfere with parasite binding mammalian cells. Structural analysis revealed that the motif TM of the nicastrin is conserved in trypanosomatids, which suggests there could be some function conservation regardless of the structural differences identified. This is the first study to describe the cellular localization, the partial structural characterization and the specific amino acid sequences involved in the immune recognition of Tc/NICT. Overall, our data should advance the understanding of the role of the y-secretase complex in the Trypanosoma cruzi.We demonstrated that


Operating ambiances alter the elemental composition of the amorphous MgFx active layer and Ti/MgFx interface region, which affects the overall device performance. The experimental results indicate that filament type resistive switching takes place at the interface of Ti/MgFx and trap-controlled space charge limited conduction (SCLC) mechanisms is dominant in both the low and high resistance states in the bulk MgFx layer. RRAM device performances at different operating ambiances are also altered by MgFx active layer treatments . Devices show the better uniformity, stability, and a higher on/off current ratio in vacuum compared to an open-air environment. The Ti/MgFx/Pt memory devices have great potential for future vacuum electronic applications.This study investigates switching characteristics of the magnesium fluoride (MgF Resistive switching random access memory (RRAM) devices are one of the emerging non-volatile memory (NVM) technologies with two terminal metal/insulator/metal (MIM) structures ,2. The sStudies show that the absorption of moisture or any gas usually happens by direct contact of the device with the environment, sidewall diffusion, and encapsulating layers. It can also happen during the device fabrication process ,7. The m2, Ta2O5, and HfO2-based devices [Several studies investigated the effects of surroundings by varying air, oxygen, and nitrogen partial pressure on RRAM devices’ performance ,12,13. S devices ,7,11,14. devices ,19,20,21To date, very few oxygen vacancy-based materials have been investigated as an active layer of RRAM devices in a vacuum. The operating ambiance heavily influences the oxygen vacancy-based active layer and the overall performance of the memory devices due to a large amount of oxygen available in the atmosphere. Exploring the alternative anion vacancy-based material, which is less influenced by the operating ambiance, can be one of the ways to overcome the limitations of oxygen vacancy-based RRAMs operating in a vacuum.x) has been utilized in many eco-friendly electronics [x, being a wide bandgap (11.3 eV) insulator, has also shown potential for RRAM devices [x/Pt devices demonstrated electroforming-free bipolar resistive switching behaviour in the open-air environment [x-based RRAM.Biodegradable magnesium fluoride  evaporation. The 50-μm-radius circular shadow mask was utilized to pattern 50-nm-thick MgFx and 150-nm-thick Ti during deposition without exposing the active layer to the open-air environment. MgFx thin films were deposited by e-beam evaporation under a base pressure of 1 × 10−6 Torr on various substrates. The evaporation rate was maintained constant at 2 Å/s. The substrate temperature was ambient.To fabricate the Ti/MgFx/Pt devices were also fabricated with a conditioned  MgFx layer. For scanning electron microscope (SEM) analysis, X-ray diffraction (XRD) analysis, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR) absorbance spectroscopy measurement, 50-nm and 1-μm-thick MgFx films were grown separately.To investigate the effect of active layer treatments, Ti/MgF−3 torr) .In an open-air laboratory environment, the electrical characteristics of the memory devices were measured using a semiconductor parameter analyzer . Voltage was applied directly to the top electrode, while the bottom electrode was grounded. The electrical characteristics of RRAM devices in a vacuum environment were measured using MS-TECH Vacuum Chamber Probe Station (<10The device fabrication process is straightforward. A minimum three different batches of samples (a batch consisting of more than twenty devices) for each sample type were analyzed to confirm the reproducibility. More than fifty devices were measured at each condition to confirm the observations and conclusions. The range of device-to-device variation was smaller than cycle-to-cycle variations because there were few process variables involved in the device fabrications and each process condition was well controlled.x/Pt device performances are mainly governed by the properties of the active MgFx layer. To understand the operating mechanisms of the devices, it is necessary to analyze the MgFx layer thoroughly. x thin films.Ti/MgFx thin films. The XRD analysis shows that the films were amorphous. The SEM images of these MgFx thin films are shown in x thin film shows the formation of small grains. Post-deposition annealed MgFx thin film also shows the formation of grains comparatively bigger than those of the as-deposited film. The crystal structure of the MgFx thin films can be manipulated from amorphous to crystalline by applying substrate temperature from ambient to 300 °C [x layer was successfully fabricated by keeping the substrate temperature at ambient temperature during e-beam deposition.o 300 °C ,25. The x films. Both films show the presence of Mg and F by the characteristic Mg 2p peak with a slight change in positions at around 52 eV. The shift in peak positions indicates the difference in the compositions. A small amount of oxygen is also found [x films found by curve-fitting and area analysis. The Mg/F ratio in the as-deposited MgFx film was around 1:1.65, which shows the existence of fluoride vacancies in the film. In contrast, the Mg/F ratio of the post-deposition annealed MgFx film was approximately 1:2.60. The increase of fluoride implies that F atoms were not necessarily missing but dislocated in interstitial sites and at grain boundaries of the amorphous film during deposition [so found ,22. Howeposition ,27. Duriposition ,27. Thusx thin-film FTIR absorbance spectra measured in the open air and vacuum environment. The stable Mg–F bond characteristic absorbance peak was found at 613 cm−1 for both the measurement conditions. However, many weak absorption peaks were observed between 3800~3500 cm−1 and 1700~1450 cm−1 in open-air measurement conditions due to the weak binding between Mg2+ sites and different vibrational modes (stretching and bending) of hydroxyl groups [2O is absorbed at the surface of the amorphous MgFx during fabrication [−1, a weak CO2 vibration band was also detected [2 are easily removed from the amorphous MgFx thin film in the vacuum condition [x active layer, as well as the Ti/MgFx interface [l groups ,29,30. Trication ,30. At aondition ,32. The nterface ,34.cc) of 0.25 mA. x-based Ti/MgFx/Pt memory device in an open-air environment. Electroforming free bipolar resistive switching behavior of the Ti/MgFx/Pt devices was observed with an on/off ratio >102. The electroforming-free characteristics is caused by the combined effects of sufficient internal fluoride vacancies and the presence of a small amount of O–H groups at the surface of the amorphous MgFx active layer [x [x/Pt devices is reported separately [SET and VREST) and currents (ILRS and IHRS) imply that filament type resistive switching takes place in Ti/MgFx/Pt devices [SET and VREST implies that the resistive switching mainly occurred at the top electrode/dielectric interface [In an open-air environment, the current-voltage (I–V) measurement was carried out for the devices with a 25 μm radius by applying double sweep DC voltage in the sequence of 0 V → +3 V → 0 V → −3 V → 0 V under the compliance current  to investigate the effects of the operating environment on the as-deposited MgFx based Ti/MgFx/Pt device performance. x/Pt memory device in a vacuum environment.I–V measurement of the devices was carried out in a vacuum (<10The first double sweep DC voltage was applied in the sequence of 0 V → +5 V → 0 V → −3 V → 0 V. The pristine device was in the high resistance state (HRS) vacuum condition.cc at electroforming voltage (VForming) around +4 V. When a negative bias voltage was applied, the device maintained in low resistance state (LRS). The I–V curve did not show any resistive switching characteristics  lower than the VForming. Similarly, the RESET process occurred at RESET voltage (VRESET) of approximately −2.5 V. The ILRS and IHRS values of the device were read at +0.50 V (VRead).When a positive bias voltage was applied, the current increased gradually as the voltage increased, and jumped sharply up to the Ix/Pt device needs an electroforming process to activate resistive switching properties and exhibiting higher initial resistance (~10 GΩ) than the initial resistance (~10 MΩ) in an atmospheric environment where the device was electroforming-free. Overall device performances changes in a vacuum with the reference of an atmospheric environment as follow: (1) Fluctuation of I–V curves decreases, (2) SET voltage slightly decreases from +1.25 V to +1.0 V, (3) RESET voltage increases from 0.9 V to 2.5 V, (4) SET and RESET current increases, (5) On/off ratio decreases from over ~103 to over 10.In a vacuum environment, the pristine Ti/MgF2 from the surface of the amorphous MgFx thin film made the active layer more resistive, and the overall initial resistance of the Ti/MgFx/Pt device increased by a factor of 3. Thus, the defect-rich amorphous MgFx layer with fluoride vacancies was insufficient to activate the resistive switching properties in the device without electroforming. The electroforming process was applied with the Icc of 0.25 mA. However, resistive switching properties were not activated, which can be attributed to the incomplete initial formation of the conduction filament (CF).In the vacuum environment, removing weakly bonded hydroxyl groups and COcc to 5 mA, the resistive switching properties of the devices were activated. With the increase of Icc, the size of the CF increased, and the resistance of the CF decreased. As a result, the ILRS increased. It was also found that IHRS and VRESET values have increased with the higher Icc due to the higher power requirement to dissolve the CF with a larger diameter [After increasing the Idiameter ,36,37.SET in a vacuum compared to the atmospheric environment [After electroforming, the CF dissolved partially during the RESET process, and the device reached new HRS, which is less resistive than the initial HRS. Further SET/RESET processes happen in the weakest point of the CF by partial reconstruction/rupture, which needed smaller Vironment .x/Pt device in the vacuum environment. The curve fittings results are shown in After electroforming, a typical I–V curve was replotted as log(I)−log(V) to investigate the conduction mechanism of the Ti/MgFn). The I–V characteristics in the negative voltage region also showed a similar pattern, but with sharp changes from HRS to LRS  and LRS (R3: 1.14) were close to 1, which indicates that ohmic conduction (I∝V) dominated in these regions. As the voltage increased from +1 V to +2 V, the slopes of HRS (R2: 3.83) gradually increased. At higher voltages, the conduction mechanism followed the Child’s law  of films. Thus, this air exposure decreased the fluorine vacancy and made the MgFx film more resistive [Amorphous MgFundaries ,28,29,30esistive . As a rex thin films could be recovered by putting it under vacuum, because water adsorption in porous MgFx thin films is a reversible [x thin-film-based Ti/MgFx/Pt memory devices mostly recover the fluorine vacancies in a vacuum environment by removing moisture. They show initial resistance similar to devices in a vacuum, a slightly higher electroforming voltage (~5 V), and an almost similar SET and RESET voltage.The reduced fluorine vacancy concentration in the air-exposed amorphous MgFversible , or at lversible , processx thin film was utilized to fabricate Ti/MgFx/Pt memory devices, and their characteristics were measured in open-air  in a vacuum environment than in open air and showed stable resistive switching memory properties. Nevertheless, a two-step electroforming process was required to stabilize the resistive switching process. The first electroforming process reduced the initial device resistance by soft breakdown to MΩ . The second electroforming process completed the formation of well-developed CF with similar VForming (~4 V) as the as-deposited device in a vacuum. x thin-film, as shown in cc of 5 mA. The further cycling experiments were carried out in the sequence of 0 V → +3 V → 0 V → −3 V → 0 V from the third cycle. Devices show very stable symmetric bipolar resistive switching properties with SET and RESET voltages around +1.5 V and −1.5 V, respectively.However, the device based on the annealed MgFx (1:1.65) becomes fluorine-rich (1:2.60) with a relatively bigger grain than as-deposited film, as shown in x thin-film-based device did not show resistive switching properties in an open-air environment.Due to the annealing, fluorine vacancy-based MgFx 1:1.5 becomesIn a vacuum, removing moisture with step-by-step soft breaking made it possible to form CF; the device showed resistive switching properties.x-based RRAM in a vacuum environment.x-based RRAM contains a small number of hydroxyl groups (moisture-related defects) at the interface of the Ti/MgFx at the initial stage in open-air. However, those moisture-related defects were removed in a vacuum. The device requires an electroforming process to form a fluorine vacancy-based CF filament at the Ti/MgFx interface of the Ti/MgFx/Pt memory device. After electroforming, the RESET and the SET processes are enabled by partial fracture and reconstruction of CF at the interface  at the interface helps the device perform electroforming-free resistive switching properties in an open-air environment. In a vacuum, the device gets more resistive due to the moisture removal and requires an electroforming process to activate its resistive switching properties.Effects of different operating ambiances (open-air and vacuum) and active layer treatments  on the performance of fluoride vacancy-based Ti/MgFx-based RRAM shows better stability with an on/off ratio > 102 in a vacuum due to the partial reversible moisture adsorption effect of MgFx. The annealed MgFx-based RRAM demonstrates symmetric bipolar resistive switching with better uniformity in vacuum due to the combined recrystallization and partial reversible moisture adsorption effect of MgFx.Overall, device performance in a vacuum is optimized by active layer treatments. The air-exposed MgF